Protein labeling provides valuable tools for studying the function of the intracellular protein such as immunostaining and immunolocalization, and developing therapeutic biologics which include poly(ethylene glycol)-protein conjugate drugs, antibody-drug conjugates (ADC), carbohydrate-protein conjugate vaccines and so on. Native protein labeling often involves the conjugation chemistry to take place at the nucleophilic side chains of the amino acids of the protein under the physiological conditions, among which lysine and cysteine are the most common labeling site. Conjugation at primary amine of the lysine residue and the N-terminus involves using acyl donors as the labeling reagents, which are listed as below.

In particular, acyl succinimidyl succinate method using NHS-ester (i.e., N-hydroxysuccinimide) is widely used strategy to label the primary amine, e.g. Lysine, as shown in the below picture (Basle, et al. Chem. Biol. 2013, 17, 213).

In the current method of protein labeling, scientists often encounter the problem of stability, solubility, and reactivity of the substrate, and the generation of by-products. For example, the amine-reactive groups like NHS-ester and anhydride are easily hydrolyzed under the physiological buffer such that the reactivity would be affected. The reaction condition is also critical to protein conjugation, wherein pH, solvents and temperature are specific for different labeling reagents. Furthermore, the byproducts (e.g., NHS) are generated after the conjugation reaction that requires further purification. Some of the by-products are even toxic to cells. In this regard, a traceless labeling method would be desired.
The aryl ortho dialdehyde, the common example as o-phthaldialdehyde (OPA), is a well-known fluorescent reagent used to detect primary amines, which has not been used previously to prepare protein conjugates. Furthermore, the existing reaction requires either the thiol or cyanide as the third reacting component and the generated product is 2H-isoindole compounds.
Wherein R and R′ represents any substituent.